Asymmetric hairpin target capture oligomers

ABSTRACT

The invention provides an improved stem-loop target capture oligomer and methods of use. Such a target capture oligomer has a target-binding segment forming a loop flanked by stem segments forming a stem. The stem segments are of unequal length. Such probes show little or no binding to immobilized probes in the absence of a target nucleic acid but offer good target sensitivity. The probes are particularly useful in multiplex methods of detection in which multiple target capture oligomers are present for detecting of multiple target nucleic acids (for example, detecting multiple polymorphic forms of a target gene).

CROSS-REFERENCE TO RELATED APPLICATIONS

The present applications is a divisional of U.S. application Ser. No.14/376,128 filed Nov. 11, 2014 now U.S. Pat. No. 10,655,165, which is aUS national stage of PCT/US2013/024499 filed Feb. 1, 2013, which claimsthe benefit of claims the benefit of U.S. application No. 61/593,829filed Feb. 1, 2012 incorporated by reference in its entirety for allpurposes.

REFERENCE TO A “SEQUENCE LISTING”

The sequence listing in file 545251SEQLST.TXT was created Apr. 5, 2020and is 7,244 bytes, which is hereby incorporated by reference in itsentirety for all purposes.

BACKGROUND

Detection of nucleic acids in a sample is useful in diagnostic,therapeutic, forensic, agricultural, food science applications and otherareas. One technique for purifying a target polynucleotide, which isoften used in diagnostic procedures, involves capturing a target nucleicacid onto a solid support. The solid support retains the target nucleicacid during one or more washing steps of the target nucleic acidpurification procedure. The captured target nucleic acid sequence can beanalyzed by various methods. One such method uses nucleic acid probesthat hybridize to a target sequence. Probes can be designed to detectdifferent target sequences such as those characteristic ofmicroorganisms, viruses, human genes, plant or animal genes, and/orpathogenic conditions. Additional analysis techniques that benefit fromcaptured target nucleic acids include amplification assays, microarrays,sequencing assays, mass spectrometry of nucleic acids.

A target nucleic acid can be captured using a target capture oligomerthat hybridizes to bind both to a target nucleic acid and to a nucleicacid fixed to a solid support. The target capture oligomer joins thetarget nucleic acid to the solid support to produce a complex comprisinga bound target nucleic acid. A labeled probe can be hybridized to thebound target and unbound labeled probe can be washed away from the solidsupport (see Stabinsky, U.S. Pat. No. 4,751,177).

A variation of a target capture oligomer has been described in which inthe absence of target the capture probe exists as a stem-loop structureand in the presence of a target nucleic acid, the target nucleic acidbinds to the loop portion, opening up the stem and making one of thearms of the loop accessible to bind an immobilized probe (see US20060068417). Such an arrangement can be useful in reducing the abilityof a target capture oligomer to hybridize with an immobilized probebefore the target capture oligomer has bound to its target nucleic acid.

SUMMARY OF THE CLAIMED INVENTION

The invention provides target capture oligomers (TACOs) comprising firstand second stem segments differing in length by at least two nucleobasesflanking a target-binding segment complementary to a target nucleicacid. Under hybridizing conditions in the absence of the target nucleicacid the target capture oligomer forms a stem-loop, intramolecularhybridization of the first and second stem segments forming the stem,and the target-binding segment forming the loop; and in the presence ofthe target nucleic acid, the target-binding segment hybridizes to thetarget nucleic acid disrupting the intramolecular hybridization of thefirst and second stem segments resulting in the first stem segment beingaccessible to hybridize to a complementary immobilized probe. In someTACOs, the first stem segment comprises at least 15 nucleobase units andthe second stem segment comprises at least five nucleobase units. Insome TACOs, the length of the second stem segment is 39-61% of thelength of the first stem segment. In some TACOs, the first stem segmentand the second stem segment differ in length by at least 5 nucleobaseunits. In some TACOs, the first and second stem segments differ inlength by at least 9 nucleobase units. In some TACOs, the first andsecond stem segments differ in length by 5-15 nucleobase units. In someTACOs, the first segment has 17-26 nucleobase units and the secondsegment has 7-16 nucleobase units. In some TACOs, the first segment has18-24 nucleobase units and the second segment has 7-15 nucleobase unitsand the first segment is at least 7 nucleobase units longer than thesecond segment. In some TACOS, the first and second stem segments occupythe 5′ and 3′ ends of the target capture oligomer respectively and thefirst stem segment is complementary to the immobilized probe. In someTACOs, the first and second stem segment comprises comprisecomplementary segments of polyA and polyT nucleobase units. In someTACOs, the first segment comprises a polyA segment and the second stemsegment comprises a polyT segment. In some TACOs, the first or secondstem segment comprises T₍₀₋₅₎A₍₁₀₋₄₀₎. In some TACOs, the first stemsegment comprises T₍₀₋₅₎A₍₁₀₋₄₀₎ and the second stem segment comprisesA₍₀₋₅₎T₍₁₀₋₄₀₎, wherein the first stem segment is longer than the secondstem segment. In some TACOs, the A₍₁₀₋₄₀₎ of the first stem segment is5-15 nucleotides longer than the T₍₁₀₋₄₀₎ of the second stem segment. Insome TACOs, the first stem segment comprises A₍₁₅₋₄₀₎ and the secondstem segment comprises T₍₁₀₋₃₀₎. In some TACOs, the target bindingsegment comprises at least one methyoxynucleobase.

The invention further provides a kit comprising a target captureoligomer as defined above and an immobilized probe immobilized probecomprising an a support bearing a probe comprising a segmentcomplementary to the first or second hairpin stem segment. In some kits,the longer of the first and second hairpin segments comprises polyA, theshorter of the first and second hairpin segments comprises polyT and theimmobilized probe segment comprises polyT, the polyT being intermediatein length between the polyA and polyT segments of the first and secondhairpin probes.

The invention further provides methods of capturing a target nucleicacid. Such methods comprise contacting a sample suspected of containingthe target nucleic acid with a target capture oligomer and animmobilized probe; the target capture oligomer comprising first andsecond hairpin stem segments differing in length by at least twonucleobase units flanking a target-binding segment complementary totarget nucleic acid, the target capture oligomer being in the form of ahairpin stem-loop, the stem being formed by intramolecular hybridizationof the first and second hairpin stem segments and the target-bindingsegment constituting the loop; the immobilized probe comprising an asupport bearing a probe comprising a segment complementary to the firstor second hairpin stem segment; wherein if the sample contains thetarget nucleic acid, the target nucleic acid hybridizes to thetarget-binding segment disrupting the intramolecular hybridization ofthe first and second hairpin stem segments, as a result of which thefirst or second hairpin segment hybridizes to the complementary segmenton the immobilized probe forming a support-bound capture hybrid; and ifthe sample does not contain the target nucleic acid the first and secondsegments remain intramolecularly hybridized as a stem. Any of the targetcapture oligomers disclosed above or elsewhere in this application canbe used in such methods.

In some methods, the target is present in the sample. Some methodsfurther comprise separating the support-bound capture hybrid from thesample. In some methods, the contacting is performed at a firsttemperature followed by a second temperature lower than the firsttemperature. In some methods, the first temperature is between themelting point of duplex formed between the first and second stemsegments and a duplex formed between the target binding segment and thetarget nucleic acid and the second temperature is below the meltingtemperature of the duplex formed form the first and second stem segmentsand a duplex formed between the second stem segment and the immobilizedprobe. In some methods, the separating is performed at the secondtemperature. Some methods further comprise releasing the target nucleicacid from the capture hybrid. Some methods further comprise detectingthe target nucleic acid. Some methods further comprise sequencing thetarget nucleic acid. In some methods the target capture oligomer is oneof a plurality of target capture oligomers having different targetbinding segments complementary to different targets. In some methods theplurality of target capture oligomers comprises at least ten targetcapture oligomers.

The invention further provides a reaction mixture comprising a targetcapture oligomer as defined in any preceding claim, an immobilized probeimmobilized probe comprising an a support bearing a probe comprising asegment complementary to the first or second hairpin stem segment and atarget nucleic acid that hybridizes to the target-binding segment of thetarget capture oligomer.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates the configuration of various target capture oligomersunder capture conditions and in the absence of a target nucleic acid towhich to hybridize. The black circle with checkered protrusionrepresents a capture bead and immobilized probe, respectively. Anasymmetrical target capture oligomer is shown in the hairpinconfiguration under such conditions, whereas a linear target captureoligomer remains in a linear configuration. Under these conditions, thefirst stem of the linear target capture oligomer is available tohybridize with the immobilized probe; however, the first stem of theasymmetric target capture oligomer is not.

FIG. 2 illustrates the various configurations of an asymmetric targetcapture oligomers under capture conditions and in the presence of atarget nucleic acid to which to hybridize. In this illustration, thereis an excess of asymmetric target capture oligomer compared to targetnucleic acid. The asymmetrical target capture oligomer bound to thetarget nucleic acid is shown in the open (non-hairpin) configuration,thereby exposing its first stem for binding with the immobilized probe.The asymmetrical target capture oligomer that is shown in the closed(hairpin) configuration has its second tem bound by its first stem, andnot to the immobilized probe.

FIG. 3 illustrates graphically the results obtained in Example 2.

FIG. 4 illustrates graphically the results obtained in Example 1.

DEFINITIONS

A nucleic acid refers to a multimeric compound comprising nucleotides oranalogs that have nitrogenous heterocyclic bases or base analogs linkedtogether to form a polymer, including conventional RNA, DNA, mixedRNA-DNA, and analogs thereof.

The nitrogenous heterocyclic bases can be referred to as nucleobaseunits. Nucleobase units can be conventional DNA or RNA bases (A, G, C,T, U), base analogs, e.g., inosine, 5-nitroindazole and others (TheBiochemistry of the Nucleic Acids 5-36, Adams et al., ed., 11th ed.,1992; van Aerschott et al., 1995, Nucl. Acids Res. 23(21): 4363-70),imidazole-4-carboxamide (Nair et al., 2001, Nucleosides NucleotidesNucl. Acids, 20(4-7):735-8), pyrimidine or purine derivatives, e.g.,modified pyrimidine base6H,8H-3,4-dihydropyrimido[4,5-c][1,2]oxazin-7-one (sometimes designated“P” base that binds A or G) and modified purine baseN6-methoxy-2,6-diaminopurine (sometimes designated “K” base that binds Cor T), hypoxanthine (Hill et al., 1998, Proc. Natl. Acad. Sci. USA95(8):4258-63, Lin and Brown, 1992, Nucl. Acids Res. 20(19):5149-52),2-amino-7-deaza-adenine (which pairs with C and T; Okamoto et al., 2002,Bioorg. Med. Chem. Lett. 12(1):97-9), N-4-methyl deoxygaunosine,4-ethyl-2′-deoxycytidine (Nguyen et al., 1998, Nucl. Acids Res.26(18):4249-58), 4,6-difluorobenzimidazole and 2,4-difluorobenzenenucleoside analogues (Kiopffer & Engels, 2005, Nucleosides NucleotidesNucl. Acids, 24(5-7) 651-4), pyrene-functionalized LNA nucleosideanalogues (Baba & Wengel, 2001, Chem. Commun. (Camb.) 20: 2114-5;Hrdlicka et al., 2005, J. Am. Chem. Soc. 127(38): 13293-9), deaza- oraza-modified purines and pyrimidines, pyrimidines with substituents atthe 5 or 6 position and purines with substituents at the 2, 6 or 8positions, 2-aminoadenine (nA), 2-thiouracil (sU),2-amino-6-methylaminopurine, O-6-methylguanine, 4-thio-pyrimidines,4-amino-pyrimidines, 4-dimethylhydrazine-pyrimidines, andO-4-alkyl-pyrimidines (U.S. Pat. No. 5,378,825; WO 93/13121; Gamper etal., 2004, Biochem. 43(31): 10224-36), and hydrophobic nucleobase unitsthat form duplex DNA without hydrogen bonding (Berger et al., 2000,Nucl. Acids Res. 28(15): 2911-4). Many derivatized and modifiednucleobase units or analogues are commercially available (e.g., GlenResearch, Sterling, Va.).

A nucleobase unit attached to a sugar, can be referred to as anucleobase unit, or monomer. Sugar moieties of a nucleic acid can beribose, deoxyribose, or similar compounds, e.g., with 2′ methoxy or 2′halide substitutions. Nucleotides and nucleosides are examples ofnucleobase units. Any of the methods and probes described herein can bepracticed with nucleotides.

The nucleobase units can be joined by a variety of linkages orconformations, including phosphodiester, phosphorothioate ormethylphosphonate linkages, peptide-nucleic acid linkages (PNA; Nielsenet al., 1994, Bioconj. Chem. 5(1): 3-7; PCT No. WO 95/32305), and alocked nucleic acid (LNA) conformation in which nucleotide monomers witha bicyclic furanose unit are locked in an RNA mimicking sugarconformation (Vester et al., 2004, Biochemistry 43(42):13233-41;Hakansson & Wengel, 2001, Bioorg. Med. Chem. Lett. 11 (7):935-8), orcombinations of such linkages in a nucleic acid strand. Nucleic acidsmay include one or more “abasic” residues, i.e., the backbone includesno nitrogenous base for one or more positions (U.S. Pat. No. 5,585,481).

A nucleic acid may include only conventional RNA or DNA sugars, basesand linkages, or may include both conventional components andsubstitutions (e.g., conventional RNA bases with 2′-O-methyl linkages,or a mixture of conventional bases and analogs). Inclusion of PNA,2′-methoxy or 2′-fluoro substituted RNA, or structures that affect theoverall charge, charge density, or steric associations of ahybridization complex, including oligomers that contain charged linkages(e.g., phosphorothioates) or neutral groups (e.g., methylphosphonates)may affect the stability of duplexes formed by nucleic acids.

Nucleic acids and their component nucleotides can exist in D or L form.The D-form is the natural form. An L-nucleic acid is the enantiomericform of a D-nucleic acid. The source of stereoisomerism in a nucleicacid resides in the sugar moiety of each monomeric unit forming thenucleic acid. Except for the stereoisomerisms at the sugar moiety ofeach monomeric unit, D and L-nucleic acids and their monomeric units areclosely analogous. Thus, for example, the sugar moieties of an L-nucleicacid can be linked to the same nucleobase units (i.e., adenine, guanine,cytosine, thymine and uracil) as occur in natural DNA or RNA, or any ofthe many known analogs of these nucleobase units. The sugar moiety ofL-nucleic acids can be ribose or deoxyribose or similar compounds (e.g.,with 2′-methodyx or 2′halide substitutions). The sugar moieties can belinked by sugar phosphodiester linkages as in D-nucleic acids or by anyof the analog linkages that have been used with D-nucleic acids, such asphosphorothioate or methylphosphonate linkages or peptide-nucleic acidlinkages.

L-nucleotides incorporating at least the conventional nucleobase units(i.e., A, C, G, T and U) are commercially available in thephosphoramidite form suitable for solid phase synthesis (e.g., ChemGenesCorporation (Wilmington, USA)). L-nucleic acids can be synthesized fromL-nucleotides using the same solid phase synthesis procedures as areused for D-nucleic acids (e.g., an ABI synthesizer and standardsynthesis protocols). L-nucleotides can also be linked to D-nucleotidesby a conventional coupling cycle (see Hauser et al., Nucleic AcidsResearch, 2006, Vol. 34, No. 18 5101-5111 (2006)), thus permittingsynthesis of a chimeric nucleic acid having one segment in D-nucleicacid form and the other in L-nucleic form.

L-nucleic acids hybridize to one another according to analogousprinciples to D-nucleic acids (e.g., by formation of Watson-Crick orHoogstein bonds) and have similar stability to hybrids of D-nucleicacids. The duplex formed from L-nucleic acids is a left-handed helixwhereas that formed from D-nucleic acids is a right handed helix.Although L-nucleic acids can hybridize to each other, as furtherillustrated by the Examples, L-nucleic acids and particularly polyA orpolyT L-nucleic acids have no ability to hybridize to a complementarysegment of a poly A or polyT D-nucleic acid.

Unless otherwise apparent from the context, reference to a nucleic acidor nucleotide without specifying whether the form is D- or L-, includeseither or both possibilities. However, the context may indicate thatonly a D nucleic acid or nucleotide is meant. For example, a nucleicacid occurring in nature would be understood to contain onlyD-nucleotides regardless whether so designated, as would a segment of aprobe that forms a stable duplex with such a nucleic acid.

An oligomer may contain a “random polymer” sequence that refers to apopulation of oligomers that are substantially the same in overalllength and other characteristics, but in which at least a portion of theoligomer is synthesized by random incorporation of different bases for aspecified length, e.g., a random assortment of all four standard bases(A, T, G, and C) in a DNA oligomer, or a random assortment of a fewbases (U and G) in a defined portion of a larger oligomer. The resultingoligomer is actually a population of oligomers whose finite number ofmembers is determined by the length and number of bases making up therandom portion (e.g., 2exp6 oligomers in a population of oligomers thatcontains a 6-nt random sequence synthesized by using 2 different bases).

Complementarity of nucleic acids means that a nucleotide sequence in onestrand of nucleic acid, due to orientation of its nucleobase groups,hydrogen bonds to another sequence on an opposing nucleic acid strand.The complementary bases typically are, in DNA, A with T and C with G,and, in RNA, C with G, and U with A. Complementarity can be perfect orsubstantial/sufficient. Perfect complementarity between two nucleicacids means that the two nucleic acids can form a duplex in which everybase in the duplex is bonded to a complementary base by Watson-Crickpairing. “Substantial” or “sufficient” complementary means that asequence in one strand is not completely and/or perfectly complementaryto a sequence in an opposing strand, but that sufficient bonding occursbetween bases on the two strands to form a stable hybrid complex in setof hybridization conditions (e.g., salt concentration and temperature).Such conditions can be predicted by using the sequences and standardmathematical calculations to predict the Tm of hybridized strands, or byempirical determination of Tm by using routine methods. Tm refers to thetemperature at which a population of hybridization complexes formedbetween two nucleic acid strands are 50% denatured. At a temperaturebelow the Tm, formation of a hybridization complex is favored, whereasat a temperature above the Tm, melting or separation of the strands inthe hybridization complex is favored. Tm may be estimated for a nucleicacid having a known G+C content in an aqueous 1 M NaCl solution byusing, e.g., Tm=81.5+0.41(% G+C), although other known Tm computationstake into account nucleic acid structural characteristics.

“Hybridization condition” refers to the cumulative environment in whichone nucleic acid strand bonds to a second nucleic acid strand bycomplementary strand interactions and hydrogen bonding to produce ahybridization complex. Such conditions include the chemical componentsand their concentrations (e.g., salts, chelating agents, formamide) ofan aqueous or organic solution containing the nucleic acids, and thetemperature of the mixture. Other factors, such as the length ofincubation time or reaction chamber dimensions may contribute to theenvironment (e.g., Sambrook et al., Molecular Cloning, A LaboratoryManual, 2.sup.nd ed., pp. 1.90-1.91, 9.47-9.51, 11.47-11.57 (Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1989)).

Specific binding of a target capture oligomer to a target nucleic ortarget nucleic acids means binding between a single defined sequence inthe first segment of a target capture oligomer and an exactly orsubstantially complementary segment on target nucleic acid(s) to form astable duplex. Such binding is detectably stronger (higher signal ormelting temperature) than binding to other nucleic acids in the samplelacking a segment exactly or substantially complementary to the singledefined target capture oligomer sequence. Non-specific binding of atarget capture oligomer to target nucleic acids means that the targetcapture oligomer can bind to a population of target sequences that donot share a segment having exact or substantial complementarity to asingle defined target capture oligomer sequence. Such can be achieved byfor example using a randomized sequence in the first segment of thecapture probe.

Lack of binding between nucleic acids can be manifested by bindingindistinguishable from nonspecific binding occurring between a randomlyselected pair of nucleic acids lacking substantial complementarity butof the same lengths as the nucleic acids in question.

“Separating” or “isolating” or “purifying” refers to removing one ormore components from a complex mixture, such as a sample. Preferably, aseparating, isolating or purifying step removes at least 70%, preferablyat least 90%, and more preferably about 95% of the target nucleic acidsfrom other sample components. A separating, isolating or purifying stepmay optionally include additional washing steps to remove non-targetsample components. At least X % refers to a range from X % to 100%inclusive of all whole and partial numbers (e.g., 70%, 82.5%, etc.)

“Release” of a capture hybrid refers to separating one or morecomponents of a capture hybrid from each other, such as separating atarget nucleic acid from a capture probe, and/or a target captureoligomer from an immobilized probe. Release of the target nucleic acidstrand separates the target from other components of a capture hybridand makes the target available for binding to a detection probe. Othercomponents of the capture hybrid may remain bound, e.g., the targetcapture oligomer strand to the immobilized probe on a capture support,without affecting target detection.

Reference to a range of value also includes integers within the rangeand subranges defined by integers in the range.

Transcription mediated amplification (TMA) is an isothermalnucleic-acid-based method that can amplify RNA or DNA targets abillion-fold in less than one hour's time. TMA technology uses twoprimers and two enzymes: RNA polymerase and reverse transcriptase. Oneprimer contains a promoter sequence for RNA polymerase. In the firststep of amplification, this primer hybridizes to the target RNA at adefined site. Reverse transcriptase creates a DNA copy of the targetrRNA by extension from the 3′ end of the promoter primer. The RNA in theresulting RNA:DNA duplex is degraded by the RNase activity of thereverse transcriptase. Next, a second primer binds to the DNA copy. Anew strand of DNA is synthesized from the end of this primer by reversetranscriptase, creating a double-stranded DNA molecule. RNA polymeraserecognizes the promoter sequence in the DNA template and initiatestranscription. Each of the newly synthesized RNA amplicons reenters theTMA process and serves as a template for a new round of replication.

Reverse-transcriptase PCR (RT-PCR) includes three major steps. The firststep is reverse transcription (RT), in which RNA is reverse transcribedto cDNA using reverse transcriptase. The RT step can be performed in thesame tube with PCR (using a temperature between 40° C. and 50° C.,depending on the properties of the reverse transcriptase used. The nextstep involves the denaturation of the dsDNA at temperature at or about95° C., so that the two strands separate and the primers can bind againat lower temperatures and begin a new chain reaction. Then, thetemperature is decreased until it reaches the annealing temperaturewhich can vary depending on the set of primers used, theirconcentration, the probe and its concentration (if used), and thecations concentration. An annealing temperature about 5° C. below thelowest Tm of the pair of primers is usually used (e.g., at or around 60°C.). RT-PCR utilizes a pair of primers, which are respectivelycomplementary to sequence on each of the two strands of the cDNA. Thefinal step of PCR amplification is DNA extension from the primers with aDNA polymerase, preferably a thermo stable taq polymerase, usually at oraround 72° C., the temperature at which the enzyme works optimally. Thelength of the incubation at each temperature, the temperaturealterations, and the number of cycles are controlled by a programmablethermal cycler.

Real-time polymerase chain reaction, also called quantitative real timepolymerase chain reaction (Q-PCR/qPCR/qrt-PCR) or kinetic polymerasechain reaction (KPCR), is a laboratory technique based on the PCR, whichis used to amplify and simultaneously quantify a targeted DNA molecule.It enables both detection and quantification (as absolute number ofcopies or relative amount when normalized to DNA input or additionalnormalizing genes) of one or more specific sequences in a DNA sample.

DETAILED DESCRIPTION

I. General

The present application provides an improved stem-loop target captureoligomer and methods of use. The stem-loop target capture oligomerdescribed in US20060068417 has the advantage of reducing binding ofempty target capture oligomers to an immobilized probe as discussed inthe Background. However, such probes may also have a disadvantage ofhaving reduced sensitivity for target detection due to the barrier ofopening up the stem-loop structure before the target nucleic acid canbind to the capture probe. The present stem-loop target captureoligomers retain the advantage of having little or no binding toimmobilized probes in the absence of a target nucleic acid but offerimprove target sensitivity. The improvement arises from having the armsof the target capture oligomer forming the stem being of unequal length.Although an understanding of mechanism is not required for practice ofthe invention, it is believed that the unequal length of the arms makesthe loop portion of the probe more accessible to binding of the targetnucleic acid thereby increasing sensitivity. The present hairpin loopprobes are particularly useful in multiplex methods of detection inwhich multiple target capture oligomers are present for detecting ofmultiple target nucleic acids (for example, detecting multiplepolymorphic forms of a target gene).

II. Target Capture Oligomers

The target capture oligomers of the invention can be subdivided into atleast three segments, a nucleic acid target binding segment flanked byfirst and second stem segments. The target-binding segment is configuredto bind to a target nucleic acid either specifically or nonspecifically(see U.S. Pat. No. 6,110,678 and WO 2008/016988). The first and secondstem segments are configured to bind to each other, and one of the stemsegments, arbitrarily designated as the first stem segment, is alsoconfigured to bind to an immobilized probe. The first and second stemsegments bind to one another by intramolecular hybridization forming astem-loop structure with the first and second stem segments forming thestem and the target-binding segment the loop. The stem-loop structure isalso sometimes referred to as a hairpin loop. In the absence of targetthe stem-loop structure forms under hybridization conditions below itsmelting temperature and the target capture oligomer can be referred toas inactive. When the target nucleic acid is present it binds to thetarget-binding loop segment separating or keeping separate the stemsegments, thus activating the target capture oligomer by allowing thefirst segment to hybridize to a complementary segment of an immobilizedprobe. Target capture oligomers can be supplied with the stem-loopstructure already formed, or with the stems separate or as mixedpopulation of molecules in some of which the stem is formed and inothers not. Regardless of the form of target captured oligomers whensupplied, the stem-loop structure can be formed in use when a targetcapture oligomer is placed under hybridization conditions below itsmelting temperature.

The target capture oligomer can be represented by the followingconfiguration: A-B-C, in which A and C are the first and second stemsegments of unequal length configured to form a double-stranded stem andB is the target-binding segment configured to form a single-strandedloop portion. In this representation either A or C can be considered the5′ end of the probe.

In some capture probes, first stem segment comprises at least 15nucleobase units and the second stem segment comprises at least 5nucleobase units. In some capture probes, the second stem segment is39-61% of the length of the first stem segment. In some capture probes,the first and second stem segments differ in length by at least 2, 3, 4,5, 6, 7, 8, 9, or 10 nucleobase units, for example a difference of 5-15nucleobase units, and preferably at least 7 nucleobase units. In somecapture probes, the first stem segment has 17-26 nucleobase units andthe second stem segment has 7-16 nucleobase units. In some captureprobes, the first stem segment has 17-25 nucleobase units and the secondstem segment 7-15 nucleobase units and the first stem segment is atleast 7 nucleobase units longer than the second stem segment. Someparticular examples of the nucleobase lengths of the first and secondstem segments forming a target capture oligomer include (listed aslength of first segment:length of second segment): (a) a range oflengths from 18:7 to 18:11; (b) a range of lengths from 21:9 to 21:12,(c) a length of 20:10, and (d) a range of lengths from 24:10 to 24:15.Ranges provided for nucleobase units are inclusive of all whole numbersmaking up the range; meaning, for example, that (a) is a first segmentthat is 18 nucleobase units in length and a second segment that is 7, 8,9, 10 or 11 nucleobase units in length. In some probes, the first stemsegment has 15-40 nucleobase units and the second stem segment 10-30nucleobase units. Preferably, the first stem segment in such captureprobes is at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleobase unitslonger, and more preferably at least 7 nucleobase units longer than thesecond stem segment.

In some capture probes, the longer of the first and second stem segmentsoccupies the 5′ end of the target capture oligomer and is configured tobind to a complementary segment on the capture probe. In other captureprobes, the longer of the first and second stem segments to occupies the3′ end of the probe and/or for the shorter of the first and second stemsegments to be configured to bind to a complementary segment of theimmobilized probe.

An example of the relative melting temperature of different duplexespresent during a target capture reaction is illustrated by discussingthe stem duplex and the target binding segment:target nucleic acidduplex. This melting temperature of a duplex formed between the firstand second stem segments is preferably lower than the meltingtemperature of the duplex formed between the target-binding segment andthe target nucleic acid. A relatively low melting temperature of thestem segment can be obtained by designing the first and second stemsegments to comprise complementary segments of polyA and polyTnucleobase units with the target binding segment and target nucleic acidduplex including at least some G and C nucleotides. In other words, ifthe first stem segment comprises polyA, the second stem segmentcomprises polyT or vice versa. Preferably, the first stem segment whichis configured to bind to a complementary segment on the immobilizedprobe comprises polyA and the second stem segment comprises polyT. ThepolyA segment of the first stem segment can then hybridize to a polyTsegment on the immobilized probe. In some probes the first segmentcomprises A15-40 (i.e., a homopolymer of 15 to 40 A's) and the secondsegment comprises T10-30 (i.e., a homopolymer of 10-30 T's). In somesuch probes, the first stem segment is at least 2, 3, 4, 5, 6, 7, 8, 9,or 10 nucleobase units longer, preferably at least 7 nucleobase unitslonger, than the second stem segment. Reverse configurations in which Tis used in place of A and vice versa are also possible.

The first stem segment as well being configured to bind the second stemsegment is configured to bind to an immobilized probe. The first stemsegment includes a nucleic acid that is substantially and preferablyexactly complementary to a nucleic acid present in the immobilizedprobes. For example, if the first segment includes a polyA homopolymer,then the nucleic acid of the immobilized probe includes a polyThomopolymer. The melting temperature of the duplex formed between thefirst stem segment and the immobilized probe is preferably less thanthat between the first stem segment and the second stem segment. Otherthings being equal, the melting temperature of the duplex formed betweenthe first and second stem segments is usually higher than that of theduplex formed between the second stem segment and immobilized probebecause the former duplex results from intramolecular hybridization andthe latter intermolecular hybridization. Differential meltingtemperatures can be additionally or alternatively be achieved by, forexample, having a shorter polyT homopolymer in the immobilized probethan in the second stem segment.

Optionally, the first and second stem segments of the immobilized probeand the complementary segment to the first stem segment in theimmobilized probe can be L-nucleic acids, as described in a co-pendingapplication PCT/US2011/052050. Because L-nucleic acids hybridize only toother L-nucleic acids, the use of L-nucleic acids can further increasethe specificity of capture of a desired target nucleic acid. For suchcapture probes, the immobilized probe also has an L-nucleic acid segmentcomplementary to the first segment of the capture probe.

The target-binding segment of the target capture oligomer is typicallydesigned to bind to a target nucleic acid sequence of interest.Optionally, the target-binding segment has 2′-O-methyl linkages or othermodified structure to enhance binding. In some capture probes, thetarget-binding segment is designed to bind to a segment within aparticular target nucleic acid and not to (or at least withsubstantially reduced affinity) other nucleic acids lacking this segmentthat are present in the sample. In other capture probes, thetarget-binding segment is designed to bind to a class of target nucleicacids (e.g., any DNA molecule) and does not necessarily substantiallydiscriminate between individual target nucleic acids within the class(e.g., by use of a randomized sequence). Excess target capture oligomeris configured so that the target binding segment does not bindnon-target species thereby activating the target capture oligomer. As aresult, excess target capture oligomer in the inactive configurationdoes not duplex with the immobilized probe causing capture ofcontaminant nucleic acids and/or cause reduced capture efficiency.

For the target-binding segment to bind to a particular target nucleicacid sequence of interest, the target-binding segment can be designed toinclude a nucleic acid that is substantially and preferably exactlycomplementary to a corresponding segment of the target nucleic acid. Thenucleic acid of such a first segment preferably includes at least 6, 10,15 or 20 nucleobase units (e.g., nucleotides). For example, the nucleicacid can contain 10-50, 10-40, 10-30 or 15-25 nucleobase units (e.g.,nucleotides) complementary to corresponding nucleotides in the targetnucleic acid. Here, as elsewhere in the application, ranges forcontiguous nucleic acid sequences are fully inclusive of all wholenumbers defining or within the range.

For a target capture oligomer to capture a population of related targetmolecules (e.g., a viral RNA population in a patient sample in whichmolecules differ from one another by the presence of mutations), thetarget-binding segment is preferably designed to be complementary to atarget segment that is relatively conserved among different members ofthe population.

For the target binding segment to bind nonspecifically to nucleic acidswithout necessarily substantially discriminating between differentsequences within a class, the target binding segment can include arandom polymer sequence made up of all four standard DNA bases (guanine(G), cytosine (C), adenine (A) and thymine (T)) or all four standard RNAbases (G, C, A, and uracil (U)) (see US 2008/0286775) The randomsequence can also include one or more base analogs (e.g., inosine,5-nitroindole) or abasic positions in the random polymer sequence. Sucha random polymer sequence can contain one or more sequences of poly-(k)bases, i.e., a random mixture of G and U or T bases (e.g., see Table 1of WIPO Handbook on Industrial Property Information and Documentation,Standard ST.25 (1998)). Sequences that include G and U/T bases can bechosen for their “wobble” property, i.e., U/T binds G or A, whereas Gbinds C or U/T. A target capture oligomer having a first segmentsynthesized with a random polymer sequence is in fact a finitepopulation of oligonucleotides that contain different random polymersequences made up of the bases included during the synthesis of therandom portion. For example, a population of nonspecific target captureoligomers that include a 15 nt random polymer sequence made up of G, C,A and T consists of 4¹⁵ members. The first segment can be designed tobind to DNA sequences preferentially relative to RNA or vice versa (seeUS 2008-0286775).

As mentioned, the melting temperature of the duplex formed thetarget-binding segment and the target nucleic acid is preferably higherthan the duplex formed between the first and second stem segments, whichis in turn preferably higher than between the immobilized probe and itscomplementary stem segment. The methods can alternatively be performedwith approximately equal melting temperatures (i.e., within a range of3° C.) or with different relative melting temperatures for any or all ofthese three duplexes The melting temperatures of duplexes can becalculated by conventional equations relating base composition andlength of a duplex to its melting temperature as discussed above.Calculation of melting temperature of a stem can also take into accountintramolecular hybridization as discussed by e.g., Markham et al.,Nucleic Acids Research, 2005, Vol. 33, Web Server issue W577-W58; seealso world wide webmfold.rit.albany.edumfold.rna.albany.edu/?q=mfold/dna-folding-form.Selection of polyA or polyT homopolymers for the stem segments of thetarget capture oligomer and the immobilized probe tends to confer alower melting temperature than that for a duplex formed betweentarget-binding segment and the nucleic acid target because the latterduplex usually also contains some C-G pairings, which confer greaterstability on a duplex than A-T pairings. A higher melting temperaturebetween the target-binding segment and the target nucleic acid allowsthe hybridization to be performed under conditions of higher stringencyin which the target capture oligomer first hybridizes to the targetnucleic acid and lower stringency in which the target capture oligomernow hybridized to the target nucleic acid hybridizes to the immobilizedprobe. When performed in this order, both target capture oligomer andtarget nucleic acid are in solution when they hybridize in whichconditions, hybridization takes place with much faster kinetics.

The target capture oligomer may or may not include additional segmentsas well as first and second stem segments and target-binding segment.For example, the nucleobase units of the stem segments and targetbinding segments can be directly connected by a phosphodiester bond (orany of the analogs thereof discussed above) or can be separated by ashort spacer or linker region, which may include nucleotides (D- or L),or other molecules, such as PEG typically found in linkers. Examples ofnon-nucleotide linkers include polysaccharides, peptides, andpolypeptides. (See e.g., WO 89/02439, and U.S. Pat. No. 5,585,481). Manydifferent non-nucleotide linkers can be used; one example being a C(9)linker. For example, a target capture oligomer can be configured A-[C(9)linker]-B-C, A-B-[C(9) linker]-C, or A-[C(9) linker]-B-[C(9) linker]-C.If a stem segment is a polyA homopolymer, the stem segment and thetarget-binding segment can be connected by one or more thymine residues.Likewise if a stem segment is a polyT homopolymer, the stem segment andthe target-binding segment can be connected by one or more adenineresidues. Thus some target capture oligomers comprise T(0-5) A(10-40)target binding segment and/or A(0-5)T(10-39) with the first segmentbeing longer than the second segment. The nomenclature A(10-40) means10-40 adenine residues, likewise T(0-5) and 0-5 thymidine residues, andso forth. If the shorter homopolymer segments in such an arrangement(e.g., A(0-5) and T(0-5) are complementary and can thus hybridize to oneanother, they can also be considered to be components of the first andsecond stem segments and taken into account in determining a meltingtemperature of a duplex formed from the first and second stem segments.

Multiple different target capture oligomers can be used in combinationin the same reaction. In this case, the different target captureoligomers typically have target-binding segment complementary todifferent target nucleic acids or different segments within the sametarget nucleic acid, and the same stem segments, so they can bindimmobilized probes having the complementary sequences to one of the stemsegments. Use of multiple different target capture oligomers can beuseful in capturing a population of related target sequences that may bepresent in a sample, for example, sequence and/or length variants. Forexample, in capturing a viral RNA population in which members differfrom one another by presence of mutations, multiple target captureoligomers binding to different conserved regions within the viral genomecan be used. The number of different target capture oligomers can be atleast 1, 2, 5, 10, 20, 50 or 100, for example, 1-100 or 2-50 or 3-25,inclusive of all whole numbers defining or within the range.

The number of target capture oligomers needed to capture a target istypically within a range of 2 nM to 20 nM (see, e.g., U.S. Pat. No.6,534,273 and e.g., Examples Section of U.S. Pat. No. 6,534,273). Insome analyses, the number of target capture oligomers in the reactionexceeds the number of available immobilized probes on magnetic beads.Excess target capture oligomers compared to immobilized probe occur formany reasons. Most commonly, the excess of target capture oligomersarises occurs when a large number of different target capture oligomersare added to a reaction to bind a variety of target nucleic acids thatmay or may not be present in a sample. Although some target captureoligomers hybridized to their targets may bind to the immobilized probe,other target capture oligomers without target nucleic acids (emptycapture probes) may also bind to the immobilized probe. The efficiencyof target nucleic acid capture begins to suffer because the immobilizedprobe becomes saturated with empty capture probes. Thus, some targetcapture oligomers bound to a nucleic acid target lack an availableimmobilized probe to which to hybridize and valuable sample is notcaptured and available for analysis.

Increasing the density of immobilized probe on a support or increasingthe number of supports in a target capture reaction is only a limitedsolution. The surface are of a support is limited so that immobilizedprobe density is finite. Also too high a concentration of supports in acapture reaction can inhibit downstream reactions, such as amplificationof captured target nucleic acid.

The present target capture oligomers that are activated by the presenceof a target nucleic acid but otherwise remains inactive in the absenceof target nucleic provide a solution. Only activated target captureoligomers are configured to hybridize an immobilized probe member.Non-activated target capture oligomers are not in a configuration forhybridizing to an immobilized probe, thereby not saturating theimmobilized probe with empty capture probes. The result is that moreimmobilized probe is available for forming a capture complex, and thatless undesired material is present in any post capture reactions orstorage environments. Capture efficiency is therefore increased withoutresorting to a corresponding increase in immobilized probe density on asupport and concentration of supports in a reaction.

The concentration of magnetic bead and target capture oligomer used fortarget capture when the captured target is subsequently subjected to areal-time detection are typically less than an otherwise similar capturereaction subjected to an end-point detection. For example, theconcentration of the target capture oligomer in the present methods canbe 5-20 pmol per reaction and the reaction volume from about 200 μl to 1ml. Without being bound by any theory, it is believed higher levels ofmagnetic bead and target capture oligomer interferes with thesensitivity of real-time detection more so than with the sensitivity ofend-point detection.

III. Immobilized Probe

An immobilized probe includes a nucleic acid joined directly orindirectly to a support. As indicated in the description of the captureprobe, the nucleic acid is substantially or preferably exactlycomplementary to a nucleic acid in the capture probe, although may ormay not be the same length (number of nucleobase units) as the nucleicacid in the capture probe. The nucleic acid in the immobilized probepreferably contains at least six contiguous nucleobase units and cancontain for example 10-45 or 10-40 or 10-30 or 10-25 or 14-25,inclusively, any range being inclusive of all whole numbers defining orwithin the range. The nucleic acid preferably includes a homopolymer,and more preferably a homopolymer of adenine or thymine. A preferredform of immobilized probe is or includes a homopolymer of 14 thymineresidues for use in combination with a target capture oligomer includinga first stem segment with a homopolymer of adenine residues. Someimmobilized probes include a homopolymeric segment with a few mismatches(e.g., at least 95% of nucleobase residues in a segment of 10-45residues are T residues). The presence of one or a small number ofmismatches can serve to decrease the melting temperature between theimmobilized probe and first stem segment below that of thetarget-binding segment and target nucleic acid (if it not already lowerwithout mismatches).

The nucleic acid moiety of an immobilized probe is typically provided insingle-stranded form, or if not, is denatured to single stranded formbefore or during use.

Any of a variety of materials may be used as a support for theimmobilized probes, e.g., matrices or particles made of nitrocellulose,nylon, glass, polyacrylate, mixed polymers, polystyrene, silanepolypropylene, and magnetically attractable materials. Monodispersemagnetic spheres are a preferred support because they are relativelyuniform in size and readily retrieved from solution by applying amagnetic force to the reaction container, preferably in an automatedsystem. An immobilized probe may be linked directly to the capturesupport, e.g., by using any of a variety of covalent linkages,chelation, or ionic interaction, or may be linked indirectly via one ormore linkers joined to the support. The linker can include one or morenucleobases of either D or L-enantiomeric forms not intended tohybridize to the target capture oligomer but to act as a spacer betweenthe nucleic acid of the immobilized probe and its support. As mentionedabove, the concentration of immobilized probe bound magnetic supportsand target capture oligomer used for target capture is typically lesswhen target capture is coupled to a real-time detection than is the casefor an end-point detection because higher concentrations of supports mayinhibit the real-time detection sensitivity. For immobilized probe boundmagnetic beads, the concentration is preferably 15-25 pg/ml, or about 20pg/ml of the target capture reaction mix.

IV. Target Nucleic Acid

A target nucleic acid refers to a nucleic acid molecule or population ofrelated nucleic acid molecules that is or may be present within asample. A target nucleic acid includes a segment (target segment) thathybridizes with the target-binding segment on the target captureoligomer to form a stable duplex. The target segment can be the same orsubstantially the same length as the nucleic acid of the target-bindingsegment of the target capture oligomer and exactly or substantiallycomplementarity to this nucleic acid. The target segment can be only asmall fraction of the total length of a target nucleic acid. Forexample, a target nucleic acid can be several thousand nucleotides longand a target segment can be for example, only 10-30 of thesenucleotides. A target nucleic acid can exist in different forms, i.e.,single-stranded, double-stranded, triple-stranded, or mixtures thereof,such as in a partially double-stranded hairpin structure or partiallydouble-stranded duplex structure, and a target segment can present onany strand (sense or anti-sense) of the structure. A target nucleic acidcan be RNA (e.g., viral RNA, micro RNA, mRNA, cRNA, rRNA, hnRNA or DNA(genomic or cDNA) among others. The target nucleic acid can be from apathogenic microorganism, such as a virus, bacteria or fungus, or can beendogenous to a patient. A target nucleic acid can be synthetic ornaturally occurring. A target nucleic acid can range in length from atleast about ten nucleotides to more than 1000 nucleotides or up to10,000 nucleotides or even greater than 10,000 nucleotides. Targetnucleic acids having 25-10,000 nucleotides are common.

Viral nucleic acids (e.g., genomic, mRNA) form a useful target foranalyses of viral sequences. Some examples of viruses that can bedetected include HIV, hepatitis (A, B, or C), herpes virus (e.g., VZV,HSV-1, HAV-6, HSV-II, CMV, and Epstein Barr virus), adenovirus, XMRV,influenza virus, flaviviruses, echovirus, rhinovirus, coxsackie virus,cornovirus, respiratory syncytial virus, mumps virus, rotavirus, measlesvirus, rubella virus, parvovirus, vaccinia virus, HTLV virus, denguevirus, MLV-related Virus, papillomavirus, molluscum virus, poliovirus,rabies virus, JC virus and arboviral encephalitis virus.

Analysis of viral nucleic acids is particularly useful for analyzingdrug resistance. Viruses mutate rapidly so that a patient is ofteninfected with a heterogeneous population of viral nucleic acids, whichchanges over time. Some of the mutations differentiating species of theheterogeneous population may be associated with resistance to a drugthat the patient has been treated with or may be treated with in thefuture. Deconvolution of the population to detect individual variantsallows detection of drug resistant mutations and their change over time,thus allowing treatment regimes to be customized to take into accountthe drug resistance of strains infecting a particular patient. Becausedrug-resistant or other mutations may present as only a small proportionof viral nucleic acid molecules, sequencing of a large number ofmolecules in the viral nucleic population may be required to provide ahigh likelihood of identifying all drug resistant mutations or at leastall, whose representation as a percentage of the total viral nucleicacid population exceeds a threshold. When the present methods ofcapturing and amplifying a target nucleic population are coupled to amassively parallel sequencing technique, at least 100,000, or 1,000,000members of the target nucleic population can be sequenced. Using thepresent methods, it is possible to identify mutations present atrepresentations of less than, for example, 10%, 1% or 0.1% can beidentified. Read lengths of for example at least 100, 500, 1000, 2000,or 5000 nucleotides of target nucleic acid can be obtained.

Human nucleic acids are useful for diagnosing diseases or susceptibilitytowards disease (e.g., cancer gene fusions, BRACA-1 or BRAC-2, p53,CFTR, cytochromes P450), for genotyping (e.g., forensic identification,paternity testing, heterozygous carrier of a gene that acts whenhomozygous, HLA typing), determining drug efficacy on an individual(e.g., companion diagnostics) and other uses.

rRNA is particularly useful for detecting and/or typing pathogenicbacteria. Examples of such bacteria include Chlamydia, rickettsialbacteria, mycobacteria, Staphylococci, treptocci, pneumonococci,meningococci and conococci, Klebsiella, Proteus, Serratia, Pseudomonas,Legionella, diphtheria, Salmonella, bacilli, cholera, tetanus, botulism,anthrax, plague, leptospirosis, Lymes disease bacteria, streptococci, orNeisseria.

The present methods are particularly useful for detecting small RNAs.For example, small RNAs (about 17-27 nt), such as microRNA (miRNA),small or short interfering RNAs (siRNA), short hairpin RNAs (shRNA), andsmall nuclear RNAs (snRNA) are difficult to separate from other samplecomponents and/or to detect by using known methods. Small RNAs are oftenrelatively rare in a biological sample which contributes to thedifficulty of their detection. Because small RNAs are importantregulatory molecules that modulate or silence gene expression via RNAinterference (RNAi), they may be important disease preventive ortherapeutic agents. Thus, the present method are useful for detectingthe presence of small RNA in biological samples to determine theirpresence, stability, therapeutic efficacy, or other characteristics in abiological sample without necessarily requiring extensive processing ornucleic acid amplification.

V. Sample

A “sample” or “biological sample” refers to any composition or mixturein which a target nucleic acid of interest may be present, includingplant or animal materials, waste materials, materials for forensicanalysis, environmental samples, and the like. A biological sampleincludes any tissue, cell, or extract derived from a living or deadorganism which may contain a target nucleic acid, e.g., peripheralblood, bone marrow, plasma, serum, biopsy tissue including lymph nodes,respiratory tissue or exudates, gastrointestinal tissue, urine, feces,semen, or other body fluids. Samples of particular interest are tissuesamples (including body fluids) from a human or an animal having orsuspected of having a disease or condition, particularly infection by avirus. Other samples of interest include industrial samples, such as forwater testing, food testing, contamination control, and the like.

Sample components may include target and non-target nucleic acids, andother materials such as salts, acids, bases, detergents, proteins,carbohydrates, lipids and other organic or inorganic materials. Thecombination of a sample with a target capture oligomer and immobilizedprobe can be referred to as a reaction mix.

A sample may or may not be subject of processing to purify or amplify atarget nucleic acid before performing the target capture assay describedbelow. It is not, for example, necessary to perform a column binding ofelution of nucleic acids. Such a step concentrates and purifies nucleicacids but also can lose a large proportion of the sample. Furtherprocessing can include simple dilution of a biological fluid with alysing solution to more complex (e.g., Su et al., J. Mol. Diagn. 2004,6:101-107; Sambrook, J. et al., 1989, Molecular Cloning, A LaboratoryManual, 2nd ed., pp. 7.37-7.57; and U.S. Pat. Nos. 5,374,522, 5,386,024,5,786,208, 5,837,452, and 6,551,778). Viral RNA samples are oftenprepared by treating plasma or serum with detergent to release RNA fromviruses. Typically, a sample containing a target nucleic acid is heatedto inactivate enzymes in the sample and to make the nucleic acids in thesample single-stranded (e.g., 90-100° C. for 2-10 min, then rapidlycooling to 0-5° C.).

VI. Target Capture Assay

A target capture assay is performed using one or more capture probes, animmobilized probe, a sample and a suitable medium to permithybridization of the target capture oligomer to the target nucleic acidand of target capture oligomer to the immobilized probe. The targetsample can be heated (e.g., to 95° C.) before performing the assay todenature any nucleic acids in double-stranded form. The components canbe mixed in any order. For example the target capture oligomer can beadded to the sample and hybridized with the target nucleic acid in thesample before adding the immobilized probe. However, for an automatedassay, it is preferable to minimize the number of adding steps bysupplying the target capture oligomer and immobilized probe at the sameor substantially the same time. In this case, the order of hybridizationcan be controlled by performing a first hybridization under conditionsin which a duplex can form between the target capture oligomer and thetarget nucleic acid but which exceeds the melting temperature of theduplex that would form between first and second stem segments of thecapture probe and between the target capture oligomer and immobilizedprobe, and then performing a second hybridization under conditions ofreduced stringency, preferably below the melting temperature of theduplexes formed between the first and second stem segments and betweenthe target capture oligomer and the immobilized probe. Stringency can bereduced by lowering the temperature of the assay mix. At the highertemperature, the target binding site duplexes with the target nucleicacid. At the lower temperature, the first and second stem segments ofcapture probes not bound to the target nucleic acid duplex with oneanother and the first stem segment of capture probes bound to the targetnucleic acid duplexes with the immobilized probe. For example, thehigher stringency hybridization can be performed at or around 60° C. andthe lower stringency hybridization by allowing cooling to roomtemperature or 25° C. Stringency can also be reduced by reducing saltconcentration or adding or increasing concentration of a chaotropicsolvent. In some methods, all steps (with the possible exception of aninitial denaturation step at higher temperature to denature doublestranded target) can be performed isothermally.

Following formation of the target nucleic acid:capture probe:immobilized probe hybrid (the capture hybrid complex) is separated wayfrom other sample components by physically separating the capturesupport using any of a variety of known methods, e.g., centrifugation,filtration, or magnetic attraction of a magnetic capture support. Theseparation is preferably performed at a temperature below the meltingtemperature of stem-loop structures formed by target capture oligomersso that empty target capture oligomers have no opportunity to denatureand thus bind to the capture probe. In some methods, the separation isperformed at a temperature less than but within 10° C. of the meltingtemperature of the stem-loop structure (e.g., at 60° C.) to maintainstringency of hybridization conditions and consequent ability todistinguished matched and unmatched target nucleic acids.

To further facilitate isolation of the target nucleic acid from othersample components that adhere non-specifically to any portion of thecapture hybrid, the capture hybrid may be washed one or more times todilute and remove other sample components. Washing may be accomplishedby dissociating the capture hybrid into its individual components in anappropriate aqueous solution (e.g., a solution containing Tris and EDTA.See e.g., U.S. Pat. No. 6,110,678) and appropriate conditions (e.g.,temperature above the Tm of the components) and then readjusting theconditions to permit reformation of the capture hybrid. However, forease of handling and minimization of steps, washing preferably rinsesthe intact capture hybrid attached to the capture support in a solutionby using conditions that maintain the capture hybrid. Preferably,capture of the target nucleic acid with washing if performed, removes atleast 70%, preferably at least 90%, and more preferably about 95% of thetarget nucleic acids from other sample components.

The target nucleic acid is then subject to PCR amplification, which inthe case of RNA samples is an RT-PCR reaction, preferably without priorrelease of the target nucleic acid from the capture complex. Although nostep is performed with intent to dissociate the target nucleic acid fromthe target capture oligomer before initiating PCR or RT-PCR, the targetnucleic acid may be partially or completely dissociated from the targetcapture oligomer in the course of thermocycling, particularly in adenaturation step performed at or around 95° C. The PCR reaction can beperformed in the same vessel (e.g., a microfuge tube) as the capturestep. The PCR reaction involves thermocycling between a high temperatureof about 95° C. (e.g., 90-99° C.) for dissociation and a low temperatureof about 60° C. e.g., 40-75, or 50-70 or 55-64° C.) for annealing.Typically, the number of complete thermocycles is at least 10, 20, 30 or40. PCR amplification is performed using one or more primer pairs. Aprimer pair used for PCR amplification includes two primerscomplementary to opposite strands of a target nucleic acid flanking theregion desired to be sequenced. For sequencing most of a viral genome(e.g., more than 50, 75 or 99%), the primers are preferably locatedclose to the ends of the viral genome. For amplification of relatedmolecules (e.g., mutant forms of the same virus present in a patientsample), the primers are preferably complementary to conserved regionsof the target nucleic acid likely to be present in most members of thepopulation. PCR amplification is described in PCR Technology: Principlesand Applications for DNA Amplification (ed. H. A. Erlich, Freeman Press,NY, N.Y., 1992); PCR Protocols: A Guide to Methods and Applications(eds. Innis, et al., Academic Press, San Diego, Calif., 1990); Mattilaet al., Nucleic Acids Res. 19, 4967 (1991); Eckert et al., PCR Methodsand Applications 1, 17 (1991); PCR (eds. McPherson et al., IRL Press,Oxford); and U.S. Pat. No. 4,683,202.

Following PCR amplification, the amplified target can optionally besubject to further processing to purify it and/or modify it to beamenable to a particularly sequencing format. Purification if desiredcan be performed on a silica column (e.g., a Qiagen gravity flowcolumn). The target nucleic acid binds to the column, where it can bewashed and then eluted. The amplified target DNA can also be adapted forsome sequencing formats by attachment of an adapter. The amplified DNAcan be tailed by Klenow-mediated addition of nucleotides (usually ahomopolymer) followed by annealing to an oligonucleotide complementaryto the added tail, and ligation. Depending on the sequencing platformused, special adaptors are ligated to the template before sequencing.Such as a SMRT bell adapter is ligated to the sample template forsequencing with a Pacific Biosciences' PacBio RS sequencer (see, e.g.,Travers et al. Nucl. Acids Res. (2010) 38 (15): e159).

The amplified target nucleic acid is suitable for sequence analysis by avariety of techniques. The capture of target nucleic acid can be coupledto several different formats of so-called next generation and thirdgeneration sequencing methods. Such methods can sequence millions oftarget templates in parallel. Such methods are particularly useful whenthe target nucleic acid is a heterogeneous mixture of variants, such asis often the case in a sample from a patient infected with a virus, suchas HIV. Among the many advantages, sequencing variants in parallelprovides a profile of drug resistant mutations in the sample, even drugmutations present in relatively minor proportions within the sample.

Some next generation sequence methods amplify by emulsion PCR. A targetnucleic acid immobilized to beads via a target capture oligomer providesa suitable starting material for emulsion PCR. The beads are mixed withPCR reagents and emulsion oil to create individual micro reactorscontaining single beads (Margulies et al., Nature 437, 376-80 (2005)).The emulsion is then broken and the individual beads with amplified DNAare sequenced. The sequencing can be pyrosequencing performed forexample using a Roche 454 GS FLX sequencer (454 Life Sciences, Branford,Conn. 06405). Alternatively, sequencing can be ligation/detectionperformed for example using an ABI SOLiD Sequencing System (LifeTechnologies, Carlsbad, Calif.A 92008). In another variation, targetnucleic acids are eluted from the target capture oligomer andimmobilized in different locations on an array (e.g., the HiScanSQ(Illumina, San Diego, Calif. 92121)). The target nucleic acids areamplified by bridge amplification and sequenced by template directedincorporation of labeled nucleotides, in an array format (Illumina). Inanother approach, target nucleic acids are eluted from the targetcapture oligomer and single molecules are analyzed by detecting inreal-time the incorporation nucleotides by a polymerase (single moleculereal time sequencing or SMRT sequencing). The nucleotides can be labelednucleotides that release a signal when incorporated (e.g., PacificBiosciences, Eid et al., Sciences 323 pp. 133-138 (2009) or unlabelednucleotides, wherein the system measures a chemical change onincorporation (e.g., Ion Torrent Personal Genome Machine (Guilform,Conn. 94080)).

Although captured target nucleic acids can be sequenced by anytechnique, third generation, next generation or massively parallelmethods offer considerable advantages over Sanger and Maxam Gilbertsequencing. Several groups have described an ultra high-throughput DNAsequencing procedure (see. e.g., Cheeseman, U.S. Pat. No. 5,302,509,Metzker et al., Nucleic Acids Res. 22: 4259 (1994)). The pyrosequencingapproach that employs four natural nucleotides (comprising a base ofadenine (A), cytosine (C), guanine (G), or thymine (T)) and severalother enzymes for sequencing DNA by synthesis is now widely used formutation detection (Ronaghi, Science 281, 363 (1998); Binladin et al.,PLoS ONE, issue 2, e197 (February 2007); Rehman et al., American Journalof Human Genetics, 86, 378 (March 2010); Lind et al., Next GenerationSequencing: The solution for high-resolution, unambiguous humanleukocyte antigen typing, Hum. Immunol. (2010), doi10.1016/jhumimm.2010.06.016 (in press); Shafer et al., J Infect Dis. 1;199(5):610 (2009)). In this approach, the detection is based on thepyrophosphate (PPi) released during the DNA polymerase reaction, thequantitative conversion of pyrophosphate to adenosine triphosphate (ATP)by sulfurylase, and the subsequent production of visible light byfirefly luciferase. More recent work performs DNA sequencing by asynthesis method mostly focused on a photocleavable chemical moiety thatis linked to a fluorescent dye to cap the 3′-OH group of deoxynucleosidetriphosphates (dNTPs) (Welch et al. Nucleosides and Nucleotides 18, 197(1999) & European Journal, 5:951-960 (1999); Xu et al., U.S. Pat. No.7,777,013; Williams et al., U.S. Pat. No. 7,645,596; Kao et al, U.S.Pat. No. 6,399,335; Nelson et al., U.S. Pat. Nos. 7,052,839 & 7,033,762;Kumar et al., U.S. Pat. No. 7,041,812; Sood et al, US Pat. App. No.2004-0152119; Eid et al., Science 323, 133 (2009)). Insequencing-by-synthesis methodology, DNA sequences are being deduced bymeasuring pyrophosphate release on testing DNA/polymerase complexes witheach deoxyribonucleotide triphosphate (dNTP) separately andsequentially. See Ronaghi et al., Science 281: 363 365 (1998); Hyman,Anal. Biochem. 174, 423 (1988); Harris, U.S. Pat. No. 7,767,400.

Sequencing platforms are further moving away from those that read aplurality of target nucleic acids towards single molecule sequencingsystems. Amplification is desirable even for single molecule sequencingschemes because target nucleic acid can be used in preparing thetemplate for sequencing. Earlier systems analyze target nucleic acids inbulk. What this means is that, for example with Sanger sequencing, aplurality of target nucleic acids are amplified in the presence ofterminating ddNTPs. Collectively, each termination position read on agel represents a plurality of amplification products that all terminatedat the same nucleobase position. Single molecule sequencing systems usenanostructures wherein the synthesis of a complementary strand ofnucleic acid from a single template is performed. These nanostructuresare typically configured to perform reads of a plurality of singlestrand nucleic acids. Each single strand contributes sequenceinformation to the sequence analysis system. See, Hardin et al., U.S.Pat. No. 7,329,492; Odera, US 2003-0190647.

For a further review of some sequencing technologies, see Cheng,Biochem. Biophys. 22: 223 227 (1995); Mardis, Annual Review of Genomicsand Human Genetics 9: 387-402 (2008) & Genome Medicine 1 (4): 40 (2009);Eid et al., Science 323, 133 (2009); Craighead et al., U.S. Pat. No.7,316,796; Lipshutz, et al., Curr. Opinion in Structural Biology., 4:376(1994); Kapranov et al., Science 296, 916 (2002); Levene et al., U.S.Pat. No. 6,917,726, Korlach et al., U.S. Pat. No. 7,056,661; Levene etal. Science 299, 682 (2003); Flusberg et al., Nature Methods v.7, no.6,p.461 (June 2010); Macevicz, U.S. Pat. Nos. 6,306,597 & 7,598,065;Balasubramanian et al., U.S. Pat. No. 7,232,656; Lapidus et al, U.S.Pat. No. 7,169,560; Rosenthal et al., U.S. Pat. No. 6,087,095; Lasken,Curr Opin. Microbiol. 10(5):510 (2007); Ronaghi et al.,Pharmacogenomics. Volume 8, 1437-41 (2007); Keating et al., PLoS One3(10):e3583 (2008); Pease et al., PNAS USA 91(11):5022 (1994); Lockhart,et al., Nat. Biotechnol. 14(13):1675 (1996); Shendure et al., Science309, 1728 (2005); Kim et al., Science 316, 1481 (2007); Valouev et al.Genome Research 18 (7): 1051 (2008); Cloonan et al., Nature Methods 5(7): 613 (2008); Tang et al. Nature Methods 6 (5): 377 (2009); McKernanet al. Genome Research 19 (9): 1527 (2009); Ecker et al., Nature ReviewsMicrobiology 6, 553 (2008).

VII. Kits

The invention also provides kits for performing the methods forcapturing and amplifying targets. Kits contain some and usually all ofat least one capture probe, at least one immobilized probe, and at leastone primer pair for PCR amplification as described above. In preferredkits, the immobilized probe is immobilized to a magnetized particle,preferably a paramagnetic bead, with homopolymeric oligomers (e.g.,polyA, polyT, polyC, or polyG) attached to it that are complementary toa homopolymeric portion of the target capture oligomer in the kit. Kitscan also include chemical compounds used in forming the capture hybridand/or detection hybrid, such as salts, buffers, chelating agents, andother inorganic or organic compounds. Kits can also include reversetranscriptase and a DNA polymerase for performing RT-PCR. Kits can alsoinclude chemicals for preparing samples for use in the invention methodswhich may include individual components or mixtures of lysing agents fordisrupting tissue or cellular material and preserving the integrity ofnucleic acids. Such compositions include enzymes, detergents, chaotropicagents, chelating agents, salts, buffering agents, and other inorganicor organic compounds. Kits can include any combination of the captureprobe, immobilize probe and primer pair components described above whichcan be packaged in combination with each other, either as a mixture orin individual containers. Kits can also contain instructions forperforming the capture methods described above.

Although the invention has been described in detail for purposes ofclarity of understanding, certain modifications may be practiced withinthe scope of the appended claims. All publications including accessionnumbers, websites and the like, and patent documents cited in thisapplication are hereby incorporated by reference in their entirety forall purposes to the same extent as if each were so individually denoted.To the extent difference version of a sequence, website or otherreference may be present at different times, the version associated withthe reference at the effective filing date is meant. The effectivefiling date means the earliest priority date at which the accessionnumber at issue is disclosed. Unless otherwise apparent from the contextany element, embodiment, step, feature or aspect of the invention can beperformed in combination with any other.

EXAMPLES Materials and Methods

Methods and reagents for nucleic acid synthesis, hybridization, anddetection of labels were used substantially as described below herein,although other routine methods and standard reagents may also be used toachieve equivalent results. Oligonucleotides were synthesized usingstandard phosphoramidite chemistry (Caruthers et al., 1987, Methods inEnzymol., 154: 287), purified using routine chromatographic methods(e.g., HPLC), and typically stored in a solution of 10 mM Tris, 1 mMEDTA (pH 7.5), at room temperature to −80° C. Transport medium generallycomprises 150 mM of hepes free acid, lithium lauryl sulfate at 294 mM or8%, ammonium sulfate at 100 mM and pH adjusted to 7.5 using lithiumhydroxide monohydrate. In the target capture steps illustrated in theexamples, magnetic particles were used as the capture support. Targetnucleic acids hybridize to the capture support using a target captureoligomer and an immobilized probe. Target capture reagent is generallymade from hepes, free acid at 250 mM, Lithium Chloride at 1.88M, EDTAfree acid at 100 mM and pH adjusted to 6.4 using Lithium Hydroxidemonohydrate. Capture support bound to target nucleic acids wereseparated from the soluble phase by applying a magnetic field to theoutside of the assay container, although those skilled in the art willappreciate that other means of separation may be used. The supernatantcontaining soluble components was removed, and the hybridizationcomplexes bound to the particles were washed (one to three times with awashing solution of sufficient ionic strength to maintain binding of thecaptured hybrid to the magnetic particles at the washing temperature,usually about 25° C.). Washing generally is performed at roomtemperature by suspending the particles in the washing solution,separating particles, and removing the supernatant, and repeating thosesteps for each wash. Amplification and real-time fluorescent detectionwere performed on the captured targets as described in the examples.

Detection assays are often designed in which multiple different targetsin a sample are to be captured and amplified. In practice, there isoften only one or a few of the multiple different targets in a sample,and/or only a small amount of some targets are present in the sample. Asa result, excess unhybridized target capture oligomers in the samplescan interfere with the capture efficiency of other targets in thatsample. One problem leading to interference of capture efficiency isthat the concentration of target capture oligomer used to capture eachdesired species of target nucleic acid in a sample results in high atotal concentration of capture oligomer. Target capture oligomers havingno captured target nucleic acid can then saturate the solid support,limiting capture of target nucleic acid from the sample. This proposedmechanism or theory about why these unhybridized target captureoligomers interfere with capture efficiency of another species is notlimiting on the current invention.

Example 1: Interference with Target Capture Efficiency by UnhybridizedTarget Capture Oligomers

To illustrate that unhybridized target capture oligomers interfere withthe efficiency of a target capture reaction, a reaction was set up tocapture an HPV target nucleic acid in the presence of linear,symmetrical hairpin and asymmetrical hairpin target capture oligomersdesigned to capture an alpha-methylacyl-co-A racemase (AMACR) targetnucleic acid. The sample includes only an HPV target nucleic acid, notan AMACR target nucleic acid. As a result, the linear, asymmetrichairpin and symmetric hairpin target capture oligomers will be in thetarget capture reactions as unhybridized target capture oligomers. Thisexample also illustrates that unhybridized asymmetrical hairpin targetcapture oligomer interferes less with capture efficiency than doesunhybridized linear capture probe.

In this example, the target nucleic acid was a HPV 35 E6/E7 in vitrotranscript (SEQ ID NO:14). Several target capture reagent mixtures weremade, each containing one of the following combinations of targetcapture oligomers: SEQ ID NOS:1 & 2; 1 & 3 or 1 & 4 or containing SEQ IDNO:1 only, each of which is described in Table 2. Also included in thetarget capture reagent mixtures was a T7 primer (SEQ ID NO:5). Severalsample mixtures were made to contain 604, 4846 or 38867 copies of atarget nucleic acid (SEQ ID NO:14) in 200 microliters of a 1:1 mixtureof water and sample transport media (Gen-Probe Incorporated, USA, Cat#301032). Target capture reactions were performed by adding 50microliters of each target capture reagent mixture described immediatelyabove to a separate sample mixture; the various reaction conditions areshown in Table 1. The target capture reaction was performed generally asfollows: a 30-min incubation at 60° C., followed by a 30-min incubationat 25° C., followed a first wash using 500 μL of wash buffer at 25° C.and then a second wash using 175 μL of wash buffer at 25° C.

Captured nucleic acids were then amplified and detected in a real-timemethod of universal transcription mediated amplification reaction (seee.g., US 2011-0003305A1). Captured target nucleic acids were each addedto a primerless amplification reagent (Gen-Probe Incorporated, USA, Cat#301032) and incubated for 15 min at 42° C. Following incubation,primers, probe (SEQ ID NOS:6-9) and enzyme reagent (Gen-Probe, Cat#301032) were added to each reaction. A series of separate amplificationreactions was then performed on each target capture condition, thereactions proceeded for 80 min at 42° C. Detection was carried out inreal-time (see e.g., U.S. Pat. No. 7,713,697 for a discussion ofreal-time detection using molecular torches). The molecular torch SEQ IDNO:6 included 2′-O-methylribonucleotides, a 9-carbon linker, a Cy5fluorescent dye and a black hole quencher (Glen Research, USA, Cat #s205932.01 and 105915-10).

TABLE 1 Reaction Conditions and Results. Copies of 10 pM of 100 pM of100 pM of 100 pM of Emergence SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID TimeNO: 14 NO: 1 NO: 2 NO: 3 NO: 4 (min) 604 Yes no No no 29.9 4846 Yes noNo no 26.9 38,867 Yes no No no 24.0 604 Yes Yes No no 32.3 4846 Yes YesNo no 28.8 38,867 Yes Yes No no 24.6 604 Yes no Yes no 29.9 4846 Yes noYes no 24.9 38,867 Yes no Yes no 23.3 604 Yes no No yes 31.0 4846 Yes noNo yes 25.4 38,867 Yes no No yes 23.9

TABLE 2 Sequences for Example 1. SEQ ID NO: Function Sequence 1HPV 35 Standard GCUCAUAACAGUAGAGAUCAGUUGUCUCTTTAAAAAAAAAAAAAAAAATCO (linear) AAAAAAAAAAAAA 2 AMACR StandardGCAGCACAUCCGACCGCUUGCTTTAAAAAAAAAAAAAAAAAAAAAAAA TCO (linear) AAAAAA 3AMACR Asymmetric TTTTTTTTTTTTTGCAGCACAUCCGACCGCUUGCAAAAAAAAAAAAAAhairpin TCO AAAAAAAAAA 4 AMACR SymmetricTTTTTTTTTTTTTTTTTTTTTTGCAGCACAUCCGACCGCUUGCAAAAA hairpin TCOAAAAAAAAAAAAAAAAA 5 T7 PrimerAATTTAATACGACTCACTATAGGGAGAGTCAGATCTACGCGCCTCACA TTTACAACAGGACG 6Molecular Torch GUCCUGUUGUAAAUGUGAGGCGAGGAC 7 Non-T7 primerGACAGCTCAGAGGAGGAGGATG 8 Primer CATCCTCCTCC Protection 9 Universal T7AATTTAATACGACTCACTATAGGGAGAGTCAGATCTACG

Emergence times are shown in Table 1 and in FIG. 4 . The presence ofunhybridized linear target capture oligomer (SEQ ID NO:2) extended theemergence time for amplification and detection of SEQ ID NO:14 (i.e.,from 29.9 to 32.3 sec for the 604 copy sample). The increased emergencetime is indicative of a lower amount of SEQ ID NO:14 being captured inthe reactions having SEQ ID NOS:1 & 2 compared to the reactions havingSEQ ID NO:1 alone. The presence of the asymmetric or symmetric hairpintarget capture oligomer (SEQ ID NOS:3 and 4 respectively) along with SEQID NO:1 interfered less or not at all with emergence times of SEQ IDNO:14 compared with SEQ ID NO:1 alone. These results show that both theasymmetric hairpin target capture oligomer and the symmetric targetcapture oligomer are useful for reducing interference with targetcapture efficiency by unhybridized probe compared to linear unhybridizedtarget capture probes.

Example 2: Comparison of Standard Linear TCO to Symmetrical andAsymmetrical Hairpin TCOs to Show Target Capture Efficiency of an IVT

The purpose of this example is to compare the sensitivity of anasymmetric hairpin target capture oligomer, a linear target captureoligomer and a symmetrical hairpin target capture oligomer. The targetnucleic acid was an AMACR in vitro transcript (SEQ ID NO:15). The targethybridizing sequences of the linear target capture oligomer (SEQ IDNO:2), the symmetrical hairpin target capture oligomer (SEQ ID NO:4 andthe asymmetrical hairpin target capture oligomer were configured tohybridize with SEQ ID NO:15. Reaction conditions were as shown in Table3.

Target capture, amplification and detection reactions were performedgenerally as described above for example 1, except that theamplification reaction was a real-time reverse transcription mediatedamplification reaction (see e.g., U.S. Pat. No. 7,374,885).Amplification and detection oligomers are shown in Table 4.

TABLE 3 Reaction Conditions and Results for Example 2. Copies of SEQ ID10 pM of 10 pM of 10 pM of Emergence NO: 15 or Urine SEQ ID SEQ ID SEQID Time Sample ID NO: 2 NO: 3 NO: 4 (min) 100 Yes No no 18.1 1000 Yes Nono 14.5 10000 Yes No no 13.0 100 no Yes no 16.7 1000 no Yes no 15.710000 no Yes no 13.6 100 no No yes 28.2 1000 no No yes 19.8 10000 no Noyes 15.9

TABLE 4 Sequences for Example 2. SEQ ID NO: Function sequence 5′ --> 3′10 Torch CUGCCAAUUUUUGAGAGAACACGGCAG 11 T7 PrimerAATTTAATACGACTCACTATAGGGAGA CCACAACGGTTTTCTGCCGGTTAGCTGGCCACGATATCAACTATTTGG 12 Non-T7 primer CCAGGAGATTCAGCGGGGCATACGGATTCTCACC 13 Blocker GCAGAAGCUUCCUGACUGGCCAAAUCC ACUCAGC

Average emergence times are provided in Table 3 and FIG. 3 . At 100copies of target nucleic acid (SEQ ID NO:15), the asymmetrical hairpintarget capture oligomer (SEQ ID NO:3) had an average emergence time ofabout 17 minutes, whereas the linear target capture oligomer (SEQ IDNO:2) and the symmetrical hairpin target capture oligomer (SEQ ID NO:4)had average emergence times of about 18 minutes and 28 minutes,respectively. Thus the capture sensitivity for the asymmetrical hairpintarget capture oligomer is at least equal to the sensitivity of a linearhairpin capture probe and much better than that of the symmetric captureprobe.

TABLE 5 In Vitro Transcripts used in the Examples SEQ ID NO:Sequence 5′->3′ Description 14CCCTATAAAAAAAACAGGGAGTGACCGAAAACGGTCGTACCGAAAACGGTTGC HPV 35 E6/E7CATAAAAGCAGAAGTGCACAAAAAAGCAGAAGTGGACAGACATTGTAAGGTGC in vitroGGTATGTTTCAGGACCCAGCTGAACGACCTTACAAACTGCATGATTTGTGCAA transcriptCGAGGTAGAAGAAAGCATCCATGAAATTTGTTTGAATTGTGTATACTGCAAACAAGAATTACAGCGGAGTGAGGTATATGACTTTGCATGCTATGATTTGTGTATAGTATATAGAGAAGGCCAGCCATATGGAGTATGCATGAAATGTTTAAAATTTTATTCAAAAATAAGTGAATATAGATGGTATAGATATAGTGTGTATGGAGAAACGTTAGAAAAACAATGCAACAAACAGTTATGTCATTTATTAATTAGGTGTATTACATGTCAAAAACCGCTGTGTCCAGTTGAAAAGCAAAGACATTTAGAAGAAAAAAAACGATTCCATAACATCGGTGGACGGTGGACAGGTCGGTGTATGTCCTGTTGGAAACCAACACGTAGAGAAACCGAGGTGTAATCATGCATGGAGAAATAACTACATTGCAAGACTATGTTTTAGATTTGGAACCCGAGGCAACTGACCTATACTGTTATGAGCAATTGTGTGACAGCTCAGAGGAGGAGGAAGATACTATTGACGGTCCAGCTGGACAAGCAAAACCAGACACCTCCAATTATAATATTGTAACGTCCTGTTGTAAATGTGAGGCGACACTACGTCTGTGTGTACAGAGCACACACATTGACATACGTAAATTGGAAGATTTATTAATGGGCACATTTGGAATAGTGTGCCCCGGCTGTTC ACAGAGAGCATAA 15GGGATTGGGAGGGCTTCTTGCAGGCTGCTGGGCTGGGGCTAAGGGCTGCTCAG AMACR IVTTTTCCTTCAGCGGGGCACTGGGAAGCGCCATGGCACTGCAGGGCATCTCGGTCGTGGAGCTGTCCGGCCTGGCCCCGGGCCCGTTCTGTGCTATGGTCCTGGCTGACTTCGGGGCGCGTGTGGTACGCGTGGACCGGCCCGGCTCCCGCTACGACGTGAGCCGCTTGGGCCGGGGCAAGCGCTCGCTAGTGCTGGACCTGAAGCAGCCGCGGGGAGCCGCCGTGCTGCGGCGTCTGTGCAAGCGGTCGGATGTGCTGCTGGAGCCCTTCCGCCGCGGTGTCATGGAGAAACTCCAGCTGGGCCCAGAGATTCTGCAGCGGGAAAATCCAAGGCTTATTTATGCCAGGCTGAGTGGATTTGGCCAGTCAGGAAGCTTCTGCCGGTTAGCTGGCCACGATATCAACTATTTGGCTTTGTCAGGTGTTCTCTCAAAAATTGGCAGAAGTGGTGAGAATCCGTATGCCCCGCTGAATCTCCTGGCTGACTTTGCTGGTGGTGGCCTTATGTGTGCACTGGGCATTATAATGGCTCTTTTTGACCGCACACGCACTGGCAAGGGTCAGGTCATTGATGCAAATATGGTGGAAGGAACAGCATATTTAAGTTCTTTTCTGTGGAAAACTCAGAAATTGAGTCTGTGGGAAGCACCTCGAGGACAGAACATGTTGGATGGTGGAGCACCTTTCTATACGACTTACAGGACAGCAGATGGGGAATTCATGGCTGTTGGAGCAATAGAACCCCAGTTCTACGAGCTGCTGATCAAAGGACTTGGACTAAAGTCTGATGAACTTCCCAATCAGATGAGCATGGATGATTGGCCAGAAATGAAGAAGAAGTTTGCAGATGTATTTGCAGAGAAGACGAAGGCAGAGTGGTGTCAAATCTTTGACGGCACAGATGCCTGTGTGACTCCGGTTCTGACTTTTGAGGAGGTTGTTCATCATGATCACAACAAGGAACGGGGCTCGTTTATCACCAGTGAGGAGCAGGACGTGAGCCCCCGCCCTGCACCTCTGCTGTTAAACACCCCAGCCATCCCTTCTTTCAAAAGGGATCCTTTCATAGGAGAACACACTGAGGAGATACTTGAAGAATTTGGATTCAGCCGCGAAGAGATTTATCAGCTTAACTCAGATAAAATCATTGAAAGTAATAAGGTAAAAGCTAGTCTCTAACTTCCAGGCCCACGGCTCAAGTGAATTTGAATACTGCATTTACAGTGTAGAGTAACACATAACATTGTATGCATGGAAACATGGAGGAACAGTATTACAGTGTCCTACCACTCTAATCAAGAAAAGAATTACAGACTCTGATTCTACAGTGATGATTGAATTCTAAAAATGGTTATCATTAGGGCTTTTGATTTATAAAACTTTGGGTACTTATACTAAATTATGGTAGTTATTCTGCCTTCCAGTTTGCTTGATATATTTGTTGATATTAAGATTCTTGACTTATATTTTGAATGGGTTCTAGTGAAAAAGGAATGATATATTCTTGAAGACATCGATATACATTTATTTACACTCTTGATTCTACAATGTAGAAAATGAGGAAATGCCACAAATTGTATGGTGATAAAAGTCACGTGAAACAGAGTGATTGGTTGCATCCAGGCCTTTTGTCTTGGTGTTCATGATCTCCCTCTAAGCACATTCCAAACTTTAGCAACAGTTATCACACTTTGTAATTTGCAAAGAAAAGTTTCACCTGTATTGAATCAGAATGCCTTCAACTGAAAAAAACATATCCAAAATAATGAGGAAATGTGTTGGCTCACTACGTAGAGTCCAGAGGGACAGTCAGTTTTAGGGTTGCCTGTATCCAGTAACTCGGGGCCTGTTTCCCCGTGGGTCTCTGGGCTGTCAGCTTTCCTTTCTCCATGTGTTTGATTTCTCCTCAGGCTGGTAGCAAGTTCTGGATCTTATACCCAACACACAGCAACATCCAGAAATAAAGATCTCAGGACCCCCCAGCAAGTCGTTTTGTGTCTCCTTGGACTGAGTTAAGTTACAAGCCTTTCTTATACCTGTCTTTGACAAAGAAGACGGGATTGTCTTTACATAAAACCAGCCTGCTCCTGGAGCTTCCCTGGACTCAACTTCCTAAAGGCATGTGAGGAAGGGGTAGATTCCACAATCTAATCCGGGTGCCATCAGAGTAGAGGGAGTAGAGAATGGATGTTGGGTAGGCCATCAATAAGGTCCATTCTGCGCAGTATCTCAACTGCCGTTCAACAATCGCAAGAGGAAGGTGGAGCAGGTTTCTTCATCTTACAGTTGAGAAAACAGAGACTCAGAAGGGCTTCTTAGTTCATGTTTCCCTTAGCGCCTCAGTGATTTTTTCATGGTGGCTTAGGCCAAAAGAAATATCTAACCATTCAATTTATAAATAATTAGGTCCCCAACGAATTAAATATTATGTCCTACCAACTTATTAGCTGCTTGAAAAATATAATACACATAAATAAAAAAA

What is claimed is:
 1. A target capture oligomer comprising a first stem segment and a second stem segment comprising complementary segments of polyA and polyT differing in length by at least five nucleobases flanking a target-binding segment complementary to a target nucleic acid, wherein under hybridizing conditions: in the absence of the target nucleic acid, the target capture oligomer forms a stem-loop structure such that, in the stem-loop structure, intramolecular hybridization of the first stem segment and the second stem segment forms the stem, and the target-binding segment forms the loop; and in the presence of the target nucleic acid, the target-binding segment hybridizes to the target nucleic acid separating or keeping separate the first stem segment and the second stem segment and resulting in the first stem segment being accessible to hybridize to a complementary immobilized probe.
 2. The target capture oligomer of claim 1, wherein the first stem segment and the second stem segment occupy the 5′ and 3′ ends of the target capture oligomer respectively and the first stem segment is complementary to the immobilized probe.
 3. The target capture oligomer of claim 1, wherein the melting temperature of a duplex formed between the target binding segment and the target nucleic acid is greater than the melting temperature of a duplex formed between the first stem segment and the second stem segment, which is greater than the melting temperature of a duplex formed between the first stem segment and the immobilized probe.
 4. The target capture oligomer of claim 1, wherein the target binding segment comprises at least one methyoxynucleobase.
 5. The target capture oligomer of claim 1, wherein the oligomer is formed by deoxyribonucleobases.
 6. A kit comprising the target capture oligomer of claim 1 and an immobilized probe comprising a support bearing a probe comprising a segment complementary to the first stem segment or the second stem segment.
 7. A reaction mixture comprising the target capture oligomer of claim 1, an immobilized probe comprising a support bearing a probe comprising a segment complementary to the first stem segment or the second stem segment and a target nucleic acid that hybridizes to the target-binding segment of the target capture oligomer. 